This invention relates to assay platforms for isolating, harvesting, detecting and/or quantitating target molecules, e.g., polypeptides, nucleotides or biomolecules, in or from a sample. More particularly, the invention relates to multiwell assay plates and other assay platforms carrying a polymer matrix having a high density of binding ligands distributed therein.
A variety of approaches and techniques have been proposed and employed to provide assay platforms for high throughput, multi-sample screening. Multiwell plates have been treated, for example, to detect binding interactions. These assay plates have a relatively low density of functional binding ligands. Consequently, the binding or capture capacities of these assay plates are at sub-microgram levels and application potential is generally restricted to detection analysis.
Solid supports made of polystyrene, polypropylene and glass, such as multiwell plates, glass slides, solid chromatography beads, sheets and tubes, are not suitable for the binding and isolation of multi-microgram amounts of high molecular weight target molecules per square centimeter including proteins, nucleic acids and polypeptides. To date no one has been able to develop on a solid support a high density, high capacity, three dimensional structure that has the appropriate architecture for binding large quantities of proteins and other molecular components. Attempts to covalently attach synthetic and natural polymers to supports have not been successful in significantly increasing the binding capacity of the supports over that obtained with passive adsorption.
The challenge of isolating and identifying total protein expressed in an organism in the rapidly growing field of proteomics requires advances in technologies such as sample preparation, purification and characterization. Current methods for isolation of proteins and other molecules require a considerable amount of effort, which generally includes employing time consuming chromatography or electrophoretic techniques. The current surface-derivatized multiwell plate systems lack sufficient surface area, porosity, and ligand density for the fast isolation of the microgram quantities needed for the characterization of proteins, nucleic acids and other biomolecules.
Attempts by others to develop a method of rapidly and specifically isolating multi-microgram amounts of proteins and other molecules per square centimeter of surface from crude cellular extracts, allowing isolation and characterization of protein, have been unsuccessful. Previous failed attempts included covalently attaching a large amount and variety of natural and synthetic molecules to flat surfaces through standard organic or photochemical means.
The present invention provides an assay platform comprising a substrate and a polymer matrix attached to the substrate, wherein the polymer matrix is capable of binding target molecules, wherein the polymer matrix comprises a plurality of polymer molecules, wherein at least some of the polymer molecules are covalently attached directly to the substrate, wherein at least some of the polymer molecules are crosslinked to other polymer molecules, wherein at least some of the polymer molecules have at least one binding ligand covalently attached thereto, and wherein the density of the polymer matrix on the substrate is at least 2 xcexcg/cm2.
The present invention also provides a method of preparing an assay platform comprising a substrate and a polymer matrix attached to the substrate, wherein the polymer matrix is capable of binding target molecules comprising:
contacting the substrate with a polymer composition comprising a plurality of polymer molecules having repeating units, wherein at least some of the polymer molecules have at least one reactive group covalently attached thereto, wherein at least some of the polymer molecules have at least one binding ligand covalently attached thereto, wherein the polymer molecules have an average molecular weight of at least 100 kDa, and wherein at least 25% of the polymer molecules have at least one reactive group and at least one binding ligand covalently attached thereto; and
activating the reactive groups to covalently bind at least some of the polymer molecules directly to the substrate and to induce cross-linking between polymer molecules to form a polymer matrix attached to the substrate.
The present invention also provides a polymer composition comprising a plurality of polymer molecules having repeating units, wherein at least some of the polymer molecules have at least one reactive group covalently attached thereto, wherein at least some of the polymer molecules have at least one binding ligand covalently attached thereto, wherein the polymer molecules have an average molecular weight of at least 100 kDa, and wherein at least 25% of polymer molecules have at least one reactive group and at least one binding ligand covalently attached thereto.